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Pathogen Diversity, Epidemiology and Control of Sclerotinia Disease in Vegetable Crops Rachel Julie Warmington A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant and Environmental Sciences The University of Warwick, School of Life Sciences September 2014 Contents List of Figures ............................................................................................................ v List of Tables............................................................................................................. xi List of Abbreviations................................................................................................ xv Acknowledgements ................................................................................................. xvi Declaration ............................................................................................................. xvii Summary .................................................................................................................... 1 1. Introduction ............................................................................................................ 2 1.1 Taxonomy of Sclerotinia species ..................................................................... 2 1.2 Host Range and Economic Importance of Sclerotinia spp. .............................. 3 1.3 Life Cycle and Epidemiology of Sclerotinia spp. ............................................ 5 1.3.1 Sclerotia and survival................................................................................. 5 1.3.2 Germination of Sclerotia, Ascospore Dispersal, Infection and Disease Development ......................................................................................... 8 1.4 Sclerotinia sclerotiorum on Carrot ................................................................. 11 1.5 Diversity of Sclerotinia sclerotiorum ............................................................. 12 1.6 Methods for Control of Sclerotinia Disease ................................................... 13 1.6.1 Plant Resistance ....................................................................................... 13 1.6.2 Fungicides ................................................................................................ 14 1.6.3 Soil Fumigation........................................................................................ 15 1.6.4 Cultural Control and Soil Solarisation ..................................................... 16 1.6.5 Soil Amendments and Biological Control ............................................... 17 1.6.6 Biofumigation .......................................................................................... 18 1.7 Project Aims and Objectives .......................................................................... 21 1.8 Thesis Structure .............................................................................................. 22 2. Frequency and diversity of Sclerotinia spp. in the UK and Norway ................... 23 2.1 Introduction .................................................................................................... 23 2.2 Materials and Methods ................................................................................... 27 2.2.1 Sampling of Sclerotinia isolates .............................................................. 27 2.2.2 Molecular Identification of Sclerotinia subarctica .................................. 29 2.2.3 Molecular Characterisation of Sclerotinia subarctica ............................. 31 2.2.4 Molecular Characterisation of Norwegian S. sclerotiorum isolates ........ 32 2.2.5 Analysis of S. sclerotiorum and S. subarctica Microsatellite Data ......... 34 i 2.2.6 Analysis of ITS and IGS Sequence Data for S. sclerotiorum and S. subarctica.......................................................................................................... 37 2.3 Results ............................................................................................................ 39 2.3.1 Molecular Identification of Sclerotinia subarctica .................................. 39 2.3.2 Molecular Characterisation of Sclerotinia subarctica Isolates ................ 39 2.3.3 Population Structure of Sclerotinia subarctica Isolates .......................... 45 2.3.4 Molecular Characterisation of Norwegian Sclerotinia sclerotiorum Isolates .............................................................................................................. 46 2.3.5 Population Structure of Norwegian Sclerotinia sclerotiorum Isolates .... 48 2.3.6 Analysis of Sequence Data for S. sclerotiorum Isolates .......................... 49 2.4 Discussion ...................................................................................................... 55 3. Susceptibility and Sclerotial Production of Sclerotinia sclerotiorum on Carrots 62 3.1 Introduction .................................................................................................... 62 3.2 Materials and Methods ................................................................................... 66 3.2.1 Carrot Root Inoculation ........................................................................... 66 3.2.2 Whole Carrot Plant Inoculation ............................................................... 69 3.2.3 Detached Carrot Leaf Inoculation............................................................ 72 3.2.4 Statistical Analyses .................................................................................. 73 3.3 Results ............................................................................................................ 75 3.3.1 Carrot Root Inoculation ........................................................................... 75 3.3.2 Whole Carrot Plant Inoculation ............................................................... 79 3.3.3 Detached Leaf Inoculation ....................................................................... 80 3.4 Discussion ...................................................................................................... 83 4. Biofumigation as a Control Measure against Sclerotinia Disease ....................... 91 4.1 Introduction .................................................................................................... 91 4.1.1 Glucosinolates and Isothiocyanates ......................................................... 91 4.1.2 Using Biofumigation against Sclerotinia Disease ................................... 94 4.2. Methods and Materials .................................................................................. 97 4.2.1 Production of Plant Material .................................................................... 97 4.2.2 HPLC Analysis of Glucosinolates ........................................................... 97 4.2.3 Production of Sclerotia ............................................................................ 99 4.2.4 Microcosm Experiments – Effect of Biofumigant Treatments on Carpogenic Germination of S. sclerotiorum Sclerotia ...................................... 99 ii 4.2.5 Microcosm Experiments – Effect of Size of Sclerotinia sclerotiorum Sclerotia on the Efficacy of Biofumigant Treatments .................................... 102 4.2.6 In vitro Experiments – Effect of Biofumigants on Mycelial Growth of Sclerotinia sclerotiorum ............................................................................. 103 4.2.7 In vitro Experiments –Effect of Biofumigant Dose on Mycelial Growth of Sclerotinia sclerotiorum ................................................................ 104 4.2.8 In vitro Experiments –Effect of Biofumigants on Carpogenic Germination of Sclerotinia sclerotiorum Sclerotia ......................................... 105 4.2.9 Plant Material Batches Used in Biofumigation Experiments ................ 107 4.2.10 Statistical Analyses .............................................................................. 108 4.3 Results .......................................................................................................... 110 4.3.1 HPLC Analysis of Glucosinolates ......................................................... 110 4.3.2 Microcosm Experiments – Effect of Biofumigant Treatments on Carpogenic Germination of S. sclerotiorum Sclerotia .................................... 115 4.3.3 Microcosm Experiments – Effect of Size of Sclerotinia sclerotiorum Sclerotia on the Efficacy of Biofumigant Treatments .................................... 118 4.3.4 In vitro Experiments – Effect of Biofumigants on Mycelial Growth of Sclerotinia sclerotiorum ............................................................................. 120 4.3.5 In vitro Experiments – Effect of Biofumigant Dose on Mycelial Growth of Sclerotinia sclerotiorum ................................................................ 122 4.3.6 In vitro Experiments – Effect of Biofumigants on Carpogenic Germination of Sclerotinia sclerotiorum Sclerotia ......................................... 124 4.4 Discussion ...................................................................................................